Inhibition of cell motility, angiogenesis, and metastasis

ABSTRACT

Disclosed are methods of inhibiting cell motility, for example, by inhibiting the binding between an intracellular transducer and a receptor protein tyrosine kinase, and more particularly by inhibiting hepatocyte growth factor (HGF) induced cell motility. The present invention also provides a method of inhibiting angiogenesis. The methods of the present invention employ peptides such as phosphotyrosyl mimetics. Also disclosed are methods of preventing and/or treating diseases, disorders, states, or conditions such as cancer, particularly metastatic cancer, for example, melanoma or prostate cancer, comprising administering to a mammal of interest one or more peptides of the present invention. Also disclosed are methods of blocking blocks HGF, VEGF, or bFGF-stimulated migration, cell proliferation, and formation of capillary-like structures.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation-in-part of co-pending U.S.patent application Ser. No. 10/111,192, which is the national phase ofPCT/US00/41423, filed Oct. 20, 2000, claiming the benefit of U.S.provisional patent application Ser. Nos. 60/160,899, filed Oct. 22, 1999and 60/221,525, filed Jul. 28, 2000, all of which are incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention in general relates to a method of inhibiting cellmotility and angiogenesis and treating various diseases in a mammal, andparticularly to a method of inhibiting cell motility and angiogenesisinduced by the hepatocyte growth factor (HGF). The present inventionalso relates to a method of blocking HGF, VEGF and bFGF-stimulated cellmigration, cell proliferation, and/or formation of capillary-likestructure. The present invention also related to a method of treatingcancer and cancer metastasis.

BACKGROUND OF THE INVENTION

The pharmaceutical industry is in search of a treatment and/orprophylaxis of proliferative diseases, disorders, or conditions such ascancers and cancer metastasis. These diseases, disorders, or conditionsaffect a large portion of the population, leading to suffering andpossibly death.

Cancer is infrequently a localized disease as cancer cells detach fromthe primary tumor, translocate to distant sites, and grow as secondarycolonies at the new anatomic locations leading to metastatic cancer. Themotility of cancer cells is associated with cancer metastasis. Theestablishment of secondary colonies also is associated with thedevelopment of new blood vessels which supply the newly formed colonywith blood and nutrients.

Development and progression of these diseases or disorders involve someform of intracellular signal transduction. Signal transduction iscritical to normal cellular homeostasis and is the process of relayingextracellular messages, e.g., chemical messages in the form of growthfactors, hormones and neurotransmitters, via receptors, e.g.,cell-surface receptors, to the interior of the cell. Protein-tyrosinekinase enzymes play a central role in this biological function.

The above enzymes catalyze the phosphorylation of specific tyrosineresidues to form tyrosine phosphorylated residues. Thetyrosine-phosphorylated proteins are involved in a range of metabolicprocesses, from proliferation and growth to differentiation. An exampleof this class of enzymes is the receptor of the hepatocyte growth factor(HGF) (also known as the scatter factor (SF)), known as c-Met. HGF is apleiotropic growth factor that, besides promoting cell survival andproliferation, has the ability to dissociate epithelial sheets and tostimulate cell motility. The dissociation of cell sheets and stimulationof cell motility is associated with the formation of new blood vessels,known as angiogenesis.

HGF stimulates mitogenesis, motogenesis, and morphogenesis in a widerange of cellular targets including epithelial and endothelial cells,hematopoietic cells, neurons, melanocytes, as well as hepatocytes. Thesepleiotropic effects play important roles during development and tissueregeneration. HGF signaling is also implicated in several human cancersincluding colon, breast, lung, thyroid, and renal carcinomas, severalsarcomas, and glioblastoma. The ability of HGF to initiate a program ofcell dissociation and increased cell motility coupled with increasedprotease production promotes aggressive cellular invasion and is linkedto tumor metastasis.

Cell dissociation and increased cell motility, such as that induced byHGF, is also associated with angiogenesis. Angiogenesis is a complex andmulti-step process that is essential for normal vascularization andwound repair. However, when the angiogenic process is not tightlyregulated, persistent and uncontrolled neovascularization occurs, whichcontributes to tumor neovascularization and cancer metastasis.

HGF signals through its cell-surface receptor. Upon HGF binding, severaltyrosine residues within the c-Met intracellular domain arephosphorylated, some of which mediate the binding of signaling proteinssuch as Grb2. Grb2 binding is involved in HGF-stimulated tubulogenesis,and is thought to link c-Met with small GTP-binding proteins such as Rhoand Rac, which are required for HGF-stimulated cytoskeletalrearrangements and cell motility. Further, VEGF and bFGF are among themost potent regulators of angiogenesis, and share intracellularsignaling mediators with a variety of angiogenesis signaling pathways.Folkman J., EXS. 79:1-8 (1997).

The foregoing indicates that there is a need for a method of inhibitingcell motility and angiogenesis. There further exists a need forinhibiting cell motility and angiogenesis induced by HGF. There furtherexists a need for inhibiting HGF, VEGF and bFGF-stimulated cellmigration, cell proliferation, and/or formation of capillary-likestructure. There further exists a need for a method of treating orpreventing diseases such as cancers and cancer metastasis in mammals.

These advantages of the present invention will be apparent from thedetailed description of the embodiments of the invention set forthbelow.

BRIEF SUMMARY OF THE INVENTION

Many of the foregoing needs have been fulfilled by the present inventionthat provides a method of inhibiting cellular motility. The presentinvention further provides a method for inhibiting angiogenesis in ananimal. A method for inhibiting the binding of intracellular transducersto receptor protein tyrosine kinases is also provided by the presentinvention. The methods of the present invention employ peptides, e.g.,phosphotyrosine mimetics, to inhibit cell motility and angiogenesis. Thepresent invention further provides methods of preventing and/or treatingdiseases, disorders, states, or conditions such as cancer, particularlymetastatic cancer. An advantage of the methods of the present inventionis that the peptides are free of cytotoxicity.

The present invention provides a method for blocking HGF-stimulatedcellular matrix invasion, a method for blocking HGF-stimulated branchingtubulogenesis, a method for blocking HGF, VEGF, or bFGF-stimulatedmigration, a method for blocking HGF, VEGF, or bFGF-stimulated cellproliferation, and a method for blocking HGF, VEGF, or bFGF-stimulatedformation of capillary structures. The phosphotyrosine mimetic peptidesdisclosed herein block HGF-stimulated matrix invasion by culturedepithelial cells or vascular endothelial cells. The peptides also blockHGF-stimulated branching tubulogenesis by cultured epithelial cells orvascular endothelial cells, e.g., those grown in a three-dimensionalextracellular matrix. The peptides also block HGF-, VEGF- andbFGF-stimulated migration by vascular endothelial cells, e.g., thosecultured in modified Boyden chambers. The peptides also block HGF-,VEGF- and bFGF-stimulated vascular endothelial cell proliferation, e.g.,in vitro. The peptides further block HGF-, VEGF- and bFGF-stimulatedformation of capillary-like structures by vascular endothelial cells,e.g., those cultured on a reconstituted extracellular matrix (Matrigel)in vitro. The peptides block in vivo angiogenesis also, as shown, e.g.,by a chick allantoid membrane assay.

The present invention further provides a method of inhibiting cancermetastasis in an animal in need thereof comprising administering aneffective amount of a Grb2-Sh2 domain binding inhibitor compound, e.g.,a compound selected from the group consisting of(N-oxalyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl) and(N-acetyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl).

While the invention has been described and disclosed below in connectionwith certain embodiments and procedures, it is not intended to limit theinvention to those specific embodiments. Rather it is intended to coverall such alternative embodiments and modifications as fall within thespirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structural formulas of peptides 1-3 that can find usein the method in accordance with embodiments of the present invention.FIG. 1 also depicts the structural formula of the control peptide 4.

FIGS. 2A and 2B depict the effect of peptides 1-3 on the migration of32D/c-Met cells. In FIGS. 2A and 2B, the x-axis represents theconcentrations of the peptides in nM and unfilled bars represent themigration of cells in the absence of HGF/NK1, and the shaded barsrepresent the migration of cells in the presence of HGF/NK1 (1 microgramper ml, final concentration). In FIG. 2A, the Y-axis represents thenumber of migrating cells. In FIG. 2B, the Y-axis represents the fold orchange in cell migration. Also included in FIGS. 2A and 2B are resultsobtained on peptide 4.

FIG. 3A depicts the effect of peptides 1-3 on the migration of Okajimacells and FIG. 3B depicts the effect of peptide 1 on the 184B5 cells. InFIGS. 3A and 3B, the X-axis represents the concentrations of thepeptides in nM; the unfilled bars represent the migration of cells inthe absence of HGF/NK1, and the shaded bars represent the migration ofcells in the presence of HGF/NK1 (300 nanograms per ml, finalconcentration). In FIG. 3A, the Y-axis represents the number ofmigrating cells. In FIG. 3B, the Y-axis represents the fold or change incell migration. Also included in FIG. 3A are the results obtained onpeptide 4.

FIG. 4 depicts photomicrographs of the effect of peptides 1-3 on thescatter of MDCK cells. Panels on the left side show cells not treatedwith HGF, and panels on the right show cells treated with HGF at 30 nM(final concentration). The peptides were added at 10 nM (finalconcentration). Also included in FIG. 4 are the results obtained onpeptide 4.

FIG. 5 depicts the effect of peptides 1, 3, and 4 on the cord length (inμm) formed by TAC-2 cells pre-treated for 18-24 hours with or withoutthe indicated concentrations of peptides 1, 3, or 4. Peptideconcentrations are indicated on the X-axis in nM. Y-axis values are meancord length per field s.e.m.

FIG. 6A depicts the effect of peptide 1 on HGF-induced HMEC-1 cellmigration. FIG. 6B depicts the effect of peptide 1 on HGF- andbFGF-induced HUVEC migration. In both FIGS. 6A and 6B, the X-axisrepresents treatment conditions. The Y-axis represents the fold increaseof HMEC-1 migration expressed as the ratio of migrating cells inHGF-treated wells (FIG. 6A) or bFGF-treated wells (FIG. 6B) to controltreated wells. In FIGS. 6A-B, unfilled bars represent the migration ofcells in the absence of HGF, while filled bars represent the migrationof cells in the presence of HGF. The gray shaded bars of FIG. 6Brepresent the migration of cells in the presence of bFGF.

FIG. 7 depicts the effect of peptide 1 on collagen matrix invasion byHMEC-1 cells. Unfilled bars represent matrix invasion by HMEC-1 cells inthe absence of HGF. Shaded bars represent matrix invasion by HMEC-1cells in the presence of HGF. The X-axis represents peptideconcentration in nM. The Y-axis represents the mean length of all thecords or single cells invading the collagen at 20 μm beneath the surfaceof the gel.

FIG. 8A depicts the effect of peptide 2 on HGF-induced HMVEC migration.FIG. 8B depicts the effect of peptide 2 on bFGF-induced HMVEC migration.In FIGS. 8A-B, the reported values are mean number of cells per opticalfield. Error bars indicate standard error of the mean (s.e.m.) of valuesfrom triplicate wells per experimental condition; where no error barsare visible, the error is too small to be shown.

FIG. 9A depicts the effect of peptide 2 on VEGF-induced cell migrationby HMEC-1 cells, FIG. 9B depicts the effect of peptide 2 on VEGF-inducedmigration by HMVE cells, FIG. 9C depicts the effect of peptide 2 onVEGF-induced cell migration by HUVE cells, and FIG. 9D depicts theeffect of peptide 1 on VEGF-induced cell migration by HUVE cells.

FIG. 10 depicts the effect of peptide 2 on PMA-induced HUVE cellmigration.

FIG. 11 depicts the effect of peptide 2 on PDGF-BB- and bFGF-inducedcell migration in NIH 3T3 fibroblasts.

FIG. 12 depicts the effect of peptide 2 on HGF-, bFGF- and VEGF-inducedHUVE and HMVE cell proliferation.

FIG. 13A depicts brightfield photomicrographs of Diff-Quick stainedcells that traversed Matrigel-coated Transwell filters (BD Biosciences)as a measure of B16 mouse melanoma cell invasiveness in vitro. The upperpanels represent the effect on HGF-treated cells (40 ng/ml, 30 h); lowerpanels depict the effect on untreated cells. The left panels representcells not treated with the SH2 domain antagonist C-90 (peptide 2); theright panels represent cells receiving increasing concentrations (nM) ofC-90 as indicated above. Objective magnification is 4×.

FIG. 13B depicts the bar graphs of PC3M cell invasion acrossMatrigel-coated Transwell filters in the presence (filled bars) orabsence (unfilled bars) of HGF (40 ng/ml, 30 h). Increasingconcentrations of C-90 (peptide 2) (nM) produces dose-dependentinhibition of PC3M cell invasiveness in vitro.

FIG. 14A depicts the imaging of C-90 (peptide 2) treated and untreatedPC3M cell metastases in the lungs ex vivo. The Y-axis units areproportional to photons emitted within a defined time interval (totalflux).

FIG. 14B depicts the imaging of primary tumor mass (left hand side) andprimary tumor volume (right hand side) of PC3M cell in the lungs forcontrol and C-90 (peptide 2) treated animals.

FIG. 14C depicts the effect on tumor metastasis in B16 mouse melanomacells receiving a single 24 h pre-treatment with C-90 (peptide 2) toform lung metastases over a three week period relative to untreatedcontrol animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for inhibiting cell motility.The present invention also provides a method for inhibiting angiogenesisin an animal. The present invention further provides a method forpreventing or treating a variety of diseases, disorders, states orconditions in a mammal, particularly in a human.

The present invention provides a method of inhibiting cell motility in amammal comprising administering to the mammal a peptide having cellsignal inhibiting activity and cell motility inhibiting activity.Advantageously, the peptide is free or substantially free ofcytotoxicity.

The present invention contemplates to retard or reduce the movement ofcells. A number of factors, forces, and/or mechanisms are involved inthe movement of cells from one location to another. The method of thepresent invention is not limited to inhibiting or interfering with oneparticular factor, force, or mechanism that is involved in the cellmovement.

The process of cell movement begins with extension of the cell membrane,the push forward of cytosol (the inner material of the cell), andretraction of the rear of the cell. As the cell membrane initially ispropelled forward, an attachment forms between the membrane and thesubstratum, thereby anchoring the “head” of the cell. Some believe thatthe cytosol is pushed forward by restructuring of the cytoskeletalnetwork within the cell, although the exact mechanism is unknown. Thefinal step involves the detachment of the “tail” of the cell from thesubstratum.

It is believed that growth factors activate a signal transductionpathway involving G-proteins, which promote cytoskeletal changesincluding actin polymerization. External factors promote cell motilityby binding to a cell surface receptor and activating a signaltransduction pathway, e.g., one involving G-proteins. The signaltransduction pathway, in turn, promotes reorganization of thecytoskeleton. A variety of extracellular factors influence cellmotility. The movement of a cell following soluble molecules along aconcentration gradient is called chemotaxis. Intracellular calcium mayplay a role in the ability of a cell to recognize concentrationgradients. Hormones such as insulin, cytokines, and specific peptidefragments of the extracellular matrix have been identified whichstimulate tumor cell motility and chemotaxis.

Aside from instigating cell motility, growth factors stimulateneovascularization, which involves, in part, cell movement. Angiogenesisbegins with proteolytic enzyme-mediated breakdown of the basementmembrane of a blood vessel. It is believed that breakdown of thebasement membrane is regulated by angiogenic factors, such as fibroblastgrowth factor. Endothelial cells migrate to the area of degradation andinvade the surrounding extracellular matrix. Invading endothelial cellsproliferate, forming an elongated column of cells. A lumen forms withinthe solid cell column, thereby forming a vessel, which eventuallyconnects with an existing blood vessel forming a capillary loop (Fotsiset al., J. Nutr., 125: 790S-797S (1995)).

The present invention provides a method for inhibiting angiogenesis inan animal, e.g., a mammal. The method comprises administering to theanimal, e.g., mammal, a peptide having cell signal inhibiting activityand cell motility inhibiting activity, wherein the peptide issubstantially free of cytotoxicity. Preferably, the peptide affectsmultiple aspects of the angiogenic process to effectivelytherapeutically or prophylactically treat angiogenesis. For example, inaddition to inhibiting cell signaling and cell motility, the peptidepreferably inhibits invasion of epithelial and/or endothelial cells intothe extracellular matrix.

In one embodiment, the present invention provides a method of inhibitingcell motility and angiogenesis induced by the hepatocyte growth factor(HGF), particularly the motility derived from a biological responsemediated by its cell surface receptor, the c-Met proto-oncogene product,a transmembrane tyrosine kinase. Upon HGF binding, several tyrosineresidues within the c-Met intracellular domain are phosphorylated. Someof the phosphorylated domains mediate binding with various signalingproteins, e.g., the Grb2 protein, the p85 subunit of phosphoinositide3-kinase (PI3K), phospholipase C-gamma, Shc, and Gab1.

Preferably, the peptide of the present inventive method is a peptidethat inhibits Grb2 SH2 domain binding. In this regard, it is imperativeto cellular function that a transducer protein accurately identifyactivated cellular receptors. Most often, recognition specificity stemsfrom the ability of the transducer protein to recognize aphosphotyrosine surrounded by a specific amino acid sequence. Therecognition motif for Grb2 is pYXN wherein pY is phospho-Tyr, X is anyamino acid, and N is Asn. Therefore, the peptide of the presentinventive method, in certain embodiments recognizes and binds a pYXNmotif. The method of the present invention is directed to inhibitingcell motility induced or mediated by signaling due to one or more of theabove HGF bindings, preferably the binding of HGF c-Met receptor withthe Grb2 protein.

The peptide employed in certain embodiments of the present invention hasthe formula I

wherein n is 0 to 15, X is a group that modifies an amino group to anamide, PTI is a bivalent radical of tyrosine, a bivalent radical ofphosphotyrosine, or of a phosphotyrosine mimetic; AA stands for abivalent radical of a natural or unnatural amino acid; and Y is asecondary amino group; or a salt thereof.

PTI in formula I above is a bivalent radical of phosphotyrosine or of aphosphotyrosine mimetic. In a preferred embodiment, n in formula I is1-4. In certain embodiments, n is 1-4 and PTI is a bivalent radical oftyrosine or a bivalent radical of phosphotyrosine or of aphosphotyrosine mimetic in the form of a bivalent radical of an aminoacid selected from the group consisting ofphosphonomethyl-phenylalanine, phosphono-α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-α-hydroxy)methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxy-phenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which can be present in the(D,L)-, D- or L-form;

-(AA)_(n)- is a bivalent radical of a tripeptide of the formula

-(AA¹)-(AA²)-(AA³)—, wherein -(AA¹)- is selected from the groupconsisting of -Ile-, -Ac₅c-, -Ac₆c-, -Asp-, -Gly-, -Phe-, -Ac₇c-, -Nbo-,-Met-, -Pro-, -β-Ala-, -Gln-, -Glu-, -DHph-, -HPh- and -tLe-; -(AA²)- isselected from the group consisting of -Asn-, -β-Ala-, -Gly-, -Ile-, and-Gln-; and -(AA³)- is selected from the group consisting of -Val-,-β-Ala-, -Gly-, -Gln-, -Asp- and Ac₅c-; a bivalent radical of adipeptide of the formula -(AA¹)-(AA²)- wherein -(AA¹)- and -(AA²)- areas recited above;

or a bivalent radical of an amino acid selected from the amino acidsmentioned above; and

Y is a monosubstituted amino selected from the group consisting of loweralkylamino, octylamino, halonaphthyloxy-lower alkylamino,naphthyloxy-lower alkylamino, phenyl-lower alkylamino, di-phenyl-loweralkylamino, (mono- or di-halo-phenyl)-lower alkylamino,naphthalenyl-lower alkylamino, hydroxy-naphthalenyl-lower alkylamino,phenanthrenyl-lower alkylamino; cycloalkylamino; and cycloalkyl-loweralkylamino;

or a salt thereof.

In some other embodiments of the present invention n is 1 to 4; PTI is abivalent radical of phosphotyrosine or of a phosphotyrosine mimetic inthe form of a bivalent radical of an amino acid selected from the groupconsisting of phosphonomethyl-phenylalanine,phosphono-(α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-(α-hydroxy)-methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxy-phenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which is present in the(D,L)-, D- or L-form;

-(AA)_(n)- is a bivalent radical of a tripeptide of the formula-(AA¹)-(AA²)-(AA³)- wherein -(AA¹)- is selected from the groupconsisting of -Ile-, -Ac₆c-, -Asp-, -Gly- and -Phe-, -(AA²)- is selectedfrom the group consisting of -Asn-, -β-Ala- and -Gly-; and -(AA³)- isselected from the group consisting of -Val-, -β-Ala-, -Gly-, -Gln-,-Asp- and -Ac₅c-;

a bivalent radical of a dipeptide of the formula -(AA¹)-(AA²)- wherein-(AA¹)- is -Ile- or -Ac₆c- and -(AA²)- is -Asn- or -β-Ala-;

or a bivalent radical of the amino acid selected from the amino acidsmentioned above; and

Y is a mono substituted amino group having a substituent selected fromthe group consisting of lower alkyl and aryl-lower alkyl;

or a salt thereof. In certain other embodiments of the presentinvention, n is 1 to 4; PTI is a bivalent radical of tyrosine or abivalent radical of phosphotyrosine mimetic in the form of a bivalentradical of an amino acid selected from the group consisting ofphosphonomethyl-phenylalanine, phosphono-α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-(α-hydroxy)methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxy-phenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which can be present in the(D,L)-, D- or the L-form;

-(AA)_(n)- is a bivalent radical of a tripeptide of the formula-(AA¹)-(AA²)-(AA³)- wherein -(AA¹)- is selected from the groupconsisting of -Ile-, -Ac₅c-, Ac₆c-, -Asp-, -Gly-, -Phe-, -Ac₇c-, -Nbo-,-Met-, -Pro-, -β-Ala-, -Gln-, -Glu-, -DHph-, -HPh- and -tLe-; -(AA²)- isselected from the group consisting of -Asn-, -β-Ala-, -Gly-, -Ile-, and-Gln-; and -(AA³)- is selected from the group consisting of -Val-,-β-Ala, -Gly-, -Gln-, -Asp- and-Ac₅c-; or

a bivalent radical of an amino acid selected from the amino acidsmentioned above; and

Y is a monosubstituted amino selected from the group consisting of loweralkylamino, octylamino, halonaphthyloxy-lower alkylamino,naphthyloxy-lower alkylamino, phenyl-lower alkylamino, di-phenyl-loweralkylamino, (mono- or di-halo-phenyl)-lower alkylamino,naphthalenyl-lower alkylamino, hydroxy-naphthalenyl-lower alkylamino orphenanthrenyl-lower alkylamino, cycloalkylamino, and cycloalkyl-loweralkylamino; or a salt thereof.

In formula I, X is a moiety attached to the nitrogen of PTI and isselected from the group consisting of C₁-C₆ alkylcarbonyl, oxalyl, C₁-C₆alkylaminooxalyl, arylaminooxalyl, aryl C₁-C₆ alkylaminooxalyl, C₁-C₆alkoxyoxalyl, carboxy C₁-C₆ alkyl carbonyl, heterocyclyl carbonyl,heterocyclyl C₁-C₆ alkyl carbonyl, aryl C₁-C₆ alkyl heterocyclyl C₁-C₆alkyl carbonyl, aryloxycarbonyl, and aryl C₁-C₆ alkoxycarbonyl. In apreferred embodiment, X is oxalyl. Particular examples of peptidesinclude oxalyl-Pmp-Ile-Asn-NH-(3-naphthalen-1-yl-propyl),oxalyl-Pmp-Ile-Asn-NH-(3-(2-hydroxy-naphthalen-1-yl)-propyl),oxalyl-Pmp-Ile-Asn-NH-(3-naphthalen-2-yl-propyl), andoxalyl-Pmp-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl) wherein “Pmp” standsfor phosphonomethyl phenylalanine.

In yet other embodiments, the peptide has the formula I, wherein PTI isa phenylalanyl radical having a phenyl ring, an amine end, and acarboxyl end, the phenyl ring having one or more substituents selectedfrom the group consisting of hydroxyl, carboxyl, formyl, carboxyalkyl,carboxyalkyloxy, dicarboxyalkyl, dicarboxyalkyloxy, dicarboxyhaloalkyl,dicarboxyhaloalkyloxy, and phosphonoalkyl, phosphonohaloalkyl, whereinthe alkyl portion of the substituents may be unsubstituted orsubstituted with a substituent selected from the group consisting ofhalo, hydroxy, carboxyl, amino, aminoalkyl, alkyl, alkoxy, and keto;

X is a moiety attached to the nitrogen of PTI and is selected from thegroup consisting of alkylcarbonyl, oxalyl, alkylaminooxalyl,arylaminooxalyl, arylalkylaminooxalyl, alkoxyoxalyl, carboxyalkylcarbonyl, heterocyclyl carbonyl, heterocyclylalkyl carbonyl, arylalkylheterocyclylalkyl carbonyl, aryloxycarbonyl, and arylalkoxycarbonyl,wherein the aryl and alkyl portions of the substituents may beunsubstituted or substituted with a substituent selected from the groupconsisting of halo, hydroxy, carboxyl, amino, aminoalkyl, alkyl, alkoxy,and keto; and the heterocyclyl portion of Y contains at least 4 heteroatoms selected from the group consisting of O, N, and S;

AA is an amino acid, the amine end of which is attached to the carboxylend of PTI; and

Y is an arylalkylamino or arylheterocyclyl alkylamino;

or a salt thereof.

Certain other embodiments of the present invention employ peptideswherein PTI is a phenylalanyl radical having a phenyl ring, an amineend, and a carboxyl end, the phenyl ring having one or more substituentsselected from the group consisting of hydroxyl, carboxyl, formyl,carboxy C₁-C₆ alkyl, carboxy C₁-C₆ alkyloxy, dicarboxy C₁-C₆ alkyl,dicarboxy C₁-C₆ alkyloxy, dicarboxyhalo C₁-C₆ alkyl, dicarboxyhalo C₁-C₆alkyloxy, and phosphono C₁-C₆ alkyl, phosphonohalo C₁-C₆ alkyl, whereinthe alkyl portion of the substituents may be unsubstituted orsubstituted with a substituent selected from the group consisting ofhalo, hydroxy, carboxyl, amino, aminoalkyl, C₁-C₆ alkyl, C₁-C₆ alkoxy,and keto;

X is a moiety attached to the nitrogen of PTI and is selected from thegroup consisting of C₁-C₆ alkylcarbonyl, oxalyl, C₁-C₆ alkylaminooxalyl,arylaminooxalyl, aryl C₁-C₆ alkylaminooxalyl, C₁-C₆ alkoxyoxalyl,carboxy C₁-C₆ alkyl carbonyl, heterocyclyl carbonyl, heterocyclyl C₁-C₆alkyl carbonyl, aryl C₁-C₆ alkyl heterocyclyl C₁-C₆ alkyl carbonyl,aryloxycarbonyl, and aryl C₁-C₆ alkoxycarbonyl, wherein the aryl andalkyl portions of the substituents may be unsubstituted or substitutedwith a substituent selected from the group consisting of halo, hydroxy,carboxyl, amino, amino C₁-C₆ alkyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, and keto;and the heterocyclyl portion of Y contains at least 4 hetero atomsselected from the group consisting of O, N, and S;

AA is an amino acid, the amine end of which is attached to the carboxylend of PTI; and

Y is an aryl C₁-C₆ alkylamino or arylheterocyclyl C₁-C₆ alkylamino;

or a salt thereof.

In any of the above embodiments, substituents can be present at anysuitable position on the phenyl ring of phenyl alanine, preferably atthe position para to the benzylic methylene group.

The peptides of formula I that can be employed in the method of thepresent invention include peptides wherein PTI is of the formula II:

wherein D has the formula III, IV, or V:

wherein R₃ and R₄ may be the same or different and are selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, aryl, aryl C₁-C₆ alkyl, C₁-C₆alkaryl, and heteroaryl; and R₅ and R₆ may be the same or different andare selected from the group consisting of hydrogen, halo, hydroxy,amino, and C₁-C₆ alkoxy; and E is selected from the group consisting ofhydrogen, C₁-C₆ alkyl, C₁-C₆ alkylcarbonyl, carboxyl, and C₁-C₆alkylcarbonyl C₁-C₆ alkyl.

A particular example of Y is aryl C₁-C₆ alkylamino. In embodiments ofpeptides used in the present inventive method, the aryl portion of Y hasthe formula:

wherein Q₁ is hydrogen or a substituent selected from the groupconsisting of hydroxyl, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, amino, andC₁-C₆ acylamino. The heteroaryl portion in certain embodiments of Y hasthe formula:

wherein Q₂ is hydrogen or a substituent selected from the groupconsisting of hydroxyl, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, amino, andC₁-C₆ acylamino, and F and G are independently selected from the groupconsisting of C, N, O, and S.

Although any suitable X can be present in the peptide, X is preferablyselected from the group consisting of acetyl, oxalyl, C₁-C₆alkylaminooxalyl, arylaminooxalyl, aryl C₁-C₆ alkylaminooxalyl, C₁-C₆alkoxyoxalyl, carboxymethylcarbonyl, tetrazolylcarbonyl,tetrazolylmethylcarbonyl, aminophenylmethoxycarbonyl, aminonaphthyloxycarbonyl, and methoxyphenylmethyl tetrazolylmethylcarbonyl,and more preferably X is oxalyl.

In certain peptides according to preferred embodiments of the presentinvention, n is 1-3.

Particular examples of peptides include(N-oxalyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl), peptide2, and (N-acetyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl),peptide 3.

Peptides having cell signaling inhibitory activity and cell motilityinhibiting activity, such as Grb2-SH2 domain mimetic peptides, areparticularly useful in inhibiting neovascularization. As demonstrated inExample 2, peptides having cell signaling inhibitory activity and cellmotility inhibiting activity, such as the Grb2-SH2 domain mimeticpeptides described herein, inhibit endothelial cell and epithelial cellinvasion of matrices and the formation of cell cords. The assays used inExample 2 mirror the angiogenic process in vivo. For instance, Matrigelis comprised of reconstituted basement membrane proteins. Cells thatinvade the Matrigel matrix form elongated cell cords, which eventuallyform interconnections (Baatout, Anticancer Research, 17: 451-456(1997)). Invasion of the extracellular matrix and the formation ofcolumns of cells therein are important processes associated withangiogenesis in vivo.

In accordance with certain embodiments, the method of the presentinvention is contemplated for use in preventing or treating variousdiseases, states, disorders, or conditions, particularly cancer.Examples of diseases, states, disorders, or conditions that arecontemplated include cancers such colon cancer, breast cancer, lungcancer, thyroid cancer, and renal cancer, sarcoma, glioblastoma, andcancer or tumor metastasis. In accordance with some embodiments, themethod of the present invention can be carried out in vitro or in vivo.

The peptides of the present invention can be prepared by methods knownto those skilled in the art. Thus, the peptides can be synthesized bythe solution phase or solid phase synthetic techniques. See, e.g., Yaoet al., J. Med. Chem., 42, 25-35 (1999); Ye et al., J. Med. Chem., 38,4270-4275 (1995); Burke, Jr. et al., Biochemistry, 33, 6490-6494 (1994);Smyth et al., Tetr. Lett. 35, 551-554 (1994); Burke, Jr. et al., J. Org.Chem., 58, 1336-1340 (1993); and Burke, Jr. et al., Tetr. Lett., 34,4125-4128 (1993).

For example, the peptides having a phosphonomethyl group on the phenylring of phenyl alanine, such as peptide 1, can be prepared by theprocedures described in U.S. patent application Ser. No. 09/236,160,filed Jan. 22, 1999, particularly in Schemes 1-4 and the Experimentalsection. The disclosure of this application is incorporated herein inits entirety by reference. Thus, for example, peptide 1 can be preparedby the reaction of t-butyl oxalyl chloride with a naphthylpropylamidotripeptide containing a phenylalanine terminal residue whose aminonitrogen is protected by a protecting group such as F-moc.

The naphthylpropylamido tripeptide can be prepared by reactingnaphthylpropylamine with N-Boc-L-Asn-N-hydroxysuccinimide ester. Theresulting naphthylpropylamido monopeptide can be further reacted with aN-protected aminocyclohexane carboxylic acid to obtain anaphthylpropylamido dipeptide. The dipeptide can then be reacted with aphosphonomethyl phenyl alanine to obtain the naphthylpropylamidotripeptide. The naphthylpropylamine can be prepared starting fromnaphthaldehyde.

As a further example, peptides having a malonyl group on the phenyl ringof phenyl alanine, such as peptide 2, can be prepared by the by theprocedures described in the provisional application Ser. No. 60/126,047,filed Mar. 23, 1999, particularly in FIGS. 1, 4-5, and 7 and Examples1-2. The disclosure of this application is incorporated herein in itsentirety by reference. Thus, for example, peptide 2 can be prepared asfollows. A naphthylpropylamido dipeptide can be prepared as above. Thedipeptide can then be reacted with a di-t-butoxy-malonylated phenylalanine whose α-amino group has been N-protected. The resultingdi-t-butoxymalonylated tripeptide can be reacted with t-butoxy oxalylchloride. The t-butoxy groups can then be cleaved off the resultingtripeptide to obtain peptide 2.

The di-t-butoxy-malonylated phenyl alanine whose α-amino group has beenN-protected can be prepared starting from p-iodotoluene by reaction withdi-t-butyl malonate. The resulting malonylated toluene derivative can behalogenated, e.g., brominated, at the methyl group to provide anα-halotoluene malonate derivative. The latter derivative can be reactedwith a benzyl-6-oxo-2,3-diphenyl-4-morpholine, and the resultingmorpholino derivative can be reduced with palladium and hydrogen toprovide a malonylated phenyl alanine. The α-amino group of this phenylalanine can be N-protected by known N-protecting groups such as F-moc.

In the practice of the method of the present invention, the peptides canbe administered as a pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective (e.g.,therapeutically or prophylactically effective) amount of at least one ofthe peptides. The pharmaceutically acceptable (e.g., pharmacologicallyacceptable) carriers described herein, for example, vehicles, adjuvants,excipients, or diluents, are well-known to those who are skilled in theart and are readily available to the public. It is preferred that thepharmaceutically acceptable carrier be one which is chemically inert tothe active compounds and one which has no detrimental side effects ortoxicity under the conditions of use.

The choice of carrier will be determined in part by the particularactive agent, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical composition of the present invention.The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intraarterial, intramuscular, interperitoneal, intrathecal,rectal, and vaginal administration are merely exemplary and are in noway limiting.

Formulations suitable for oral administration can comprise (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations can include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant, suspending agent, or emulsifying agent. Capsule forms can beof the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like containing, in addition to the active ingredient, suchcarriers as are known in the art.

The peptides, alone or in combination with other suitable components,can be made into aerosol formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also can be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol,glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers,such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acidester or glyceride, or an acetylated fatty acid glyceride with orwithout the addition of a pharmaceutically acceptable surfactant, suchas a soap or a detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyl dialkyl ammoniumhalides, and alkyl pyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylenepolypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl-β-aminopropionates, and 2-alkyl-imidazolinequaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants. The quantityof surfactant in such formulations typically ranges from about 5 toabout 15% by weight. Suitable surfactants include polyethylene sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The peptides or derivatives thereof may also be made into injectableformulations. The requirements for effective pharmaceutical carriers forinjectable compositions are well known to those of ordinary skill in theart. See, e.g., Pharmaceutics and Pharmacy Practice, J. B. LippincottCo., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982),and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986).

Additionally, the peptides of the present invention may be made intosuppositories by mixing with a variety of bases, such as emulsifyingbases or water-soluble bases. Formulations suitable for vaginaladministration may be presented as pessaries, tampons, creams, gels,pastes, foams, or spray formulas containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day if desired. Inproper doses and with suitable administration of certain compounds, thepresent invention provides for a wide range of responses. Typically thedosages range from about 0.001 to about 1000 mg/kg body weight of theanimal being treated/day. Preferred dosages range from about 0.01 toabout 10 mg/kg body weight/day, and further preferred dosages range fromabout 0.01 to about 1 mg/kg body weight/day.

The effects of the methods of the present invention can be determined byany suitable methods, such as methods known to those skilled in the art.For example, the present invention provides a method of inhibiting, inwhole or in part, angiogenesis. The ordinarily skilled artisan has theability to detect inhibition of angiogenesis using a variety of methods,such as, for example, fluorescein angiography, scanning electronmicroscopy, and generation of vascular casts. In addition, severalanimal models of angiogenesis exist including, but not limited to, themouse ear model of neovascularization, models of ocularneovascularization in rabbits, and the rat hindlimb ischemia model ofneovascularization. In the treatment of cancer, the change in tumor sizecan be measured, e.g., by imaging techniques, at suitable intervalsduring the treatment period as well as after the treatment isdiscontinued. Alternatively, biological fluid samples, e.g., bloodsamples can be drawn at predetermined intervals to determine theconcentration of cancer cells therein. Biopsy can be carried out todetermine the characteristics of tumor cells. The peptides of thepresent invention are contemplated for use in the prevention ofdiseases. Thus, a disease, e.g., metastasis of cancer, is contemplatedto be prevented in whole or in part.

The peptides of the present invention have the advantage that they arestable to or in presence of enzymes encountered during in vivo use. Thepeptides can find use in in vitro and in vivo applications. For example,they can find use as molecular probes as well as in assays to identify,isolate, and/or quantitate receptor or binding sites in a cell ortissue. The peptides also can find use in vivo for studying the efficacyin the treatment of various diseases or conditions involving SH2domains.

The present invention further provides a method for inhibiting thebinding between an intracellular transducer and a receptor proteintyrosine kinase that influences cell motility comprising contacting thereceptor with a peptide of the present invention, or an ester or etherderivative thereof. An example of an intracellular transducer is onethat includes one or more SH2 domains, preferably the Grb2 transducer.An example of a receptor protein tyrosine kinase is the HGF factor,particularly the HGF/c-Met receptor.

The peptides of the present invention interact with intracellular signaltransducers, thus interfering in the pathways leading to cellproliferation and movement. These biological effects can be utilized toinhibit growth of neoplastic cells, inhibit angiogenesis, and to preventmetastatic spreading. The present invention provides a method forpreventing or treating a disease, condition, or state in a mammal thatis mediated by the binding of an intracellular transducer to a receptorprotein tyrosine kinase comprising administering to the mammal a peptideof the present invention.

The peptides of the present invention can be used to prevent and/ortreat a disease, disorder, state, or condition such as cancer. Examplesof cancers that may be prevented or treated include, but are not limitedto, colon cancer, breast cancer, lung cancer, thyroid cancer, and renalcancer. Further examples of disease, disorder, state, or condition thatcan be prevented or treated include sarcoma, lymphoma, melanoma,leukemia, glioblastoma, and tumor metastasis, for example, metastasis ofmelanoma or prostate tumor.

The present invention further provides a method for inhibiting thebinding of an intracellular transducer to a receptor protein tyrosinekinase comprising contacting (a) a sample containing the receptorprotein tyrosine kinase, (b) the intracellular transducer, and (c) thepeptide of the present invention, under conditions wherein, in theabsence of the peptide, the receptor protein tyrosine kinase binds tothe intracellular transducer; wherein the contacting results in theinhibition of binding of the intracellular transducer to the receptorprotein tyrosine kinase.

The present invention further provides a method for detecting theinhibition of binding of an intracellular transducer to a receptorprotein tyrosine kinase comprising (a) contacting a sample containingthe receptor protein tyrosine kinase with the intracellular transducer,and separately, in the presence and absence of the peptide of thepresent invention or a derivative thereof, under conditions that allowfor binding of the receptor protein tyrosine kinase to the intracellulartransducer in the absence of the peptide; (b) detecting whether bindinghas occurred between the receptor protein tyrosine kinase and theintracellular transducer; and (c) comparing relative binding levels ofthe receptor protein tyrosine kinase to the intracellular transducer inthe presence and absence of the peptide; wherein the detection ofdecreased binding in the presence of the peptide indicates inhibition ofbinding.

The present invention further provides a method for determining thepresence of a Grb2 protein in a material comprising (a) exposing asample of the material to a Grb2 binding compound and obtaining a firstbinding result; (b) exposing another sample of the material to a peptideof the present invention, or a derivative thereof, and obtaining asecond binding result; and (c) comparing the first and second bindingresults to determine whether Grb2 protein is present in the material.

The peptides of the present invention inhibit cell motility. Thepeptides prevent scattering of cells.

The cytotoxic effects of agents that disrupt the cytoskeleton, such ascolchicine, taxol, cytochalasins, and phalloidin are well-characterized,and are fundamentally different from the anti-motility effects exertedby the peptides employed in the present invention. These peptides may behighly efficacious for the safe treatment of human diseases such asmetastatic cancers, e.g., where the role of HGF plays a role instimulating the invasion of cells into tissue surrounding the tumors andthe migration of metastases to distant sites.

In an embodiment, the invention provides a method of inhibiting cancermetastasis in an animal in need thereof comprising administering aneffective amount of a compound selected from the group consisting of(N-oxalyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl) and(N-acetyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl). Thecancer can be, for example, melanoma or prostate cancer.

EXAMPLES

The following examples further illustrate the present invention but, ofcourse, should not be construed as in any way limiting its scope.

Example 1

This Example illustrates the inhibition of cell motility in accordancewith a method of the present invention.

Materials

HGF/NK1 was produced in a bacterial expression system, purified andrefolded as described in Stahl et al., Biochem. J., 326: 763-772 (1997).Peptides 1-4 were synthesized and purified as described in Yao et al.,supra. The Grb2 binding properties of these compounds in vitro have beendescribed previously (Yao et al. supra). Among these peptides, 4 has thelowest affinity for Grb2 by at least 100-fold. Accordingly, this peptidewas used as a negative control in the biological experiments discussedherein.

MDCK Cell Scatter Assay

MDCK cell movement, observed as the dispersion or scatter of singlecells from tightly grouped colonies, was assayed as described in Stokeret al., J. Cell Sci. 77, 209-223 (1985). Briefly, MDCK cells were seededat the final density of 2×10⁴ cells/well into 24 well plates in DMEMcontaining various concentrations of inhibitors. Four hours later HGF(30 ng/ml) was added, and cells were incubated for and additional 16 hat 37° C. The scatter of fixed and stained cells was observed by lightmicroscopy.

Cell Migration Assay

Migration by 185B5 and Okajima cells was measured using Biocoat CellEnvironments control inserts (8 micron pore size; Becton Dickinson). Thelower chamber contained RPMI+0.1% FBS, to which growth factors orinhibitors were added. Cells were trypsinized, washed in RPMI+0.1% FBS,added to the upper chamber at a final density of 2×10⁵ cells/ml, andincubated for 16 h at 37° C. Cells were fixed and stained usingDiff-Quik (Dade Diagnostics of P.R. Inc.), and number of cells that hadtraversed the membrane were counted using low-power brightfieldmicroscopy. The difference in this number between untreated and treatedcells is designated on the y-axis as “Fold Increase” in migration.32D/c-Met cell migration was assayed using a modified Boyden chamberwith 5 micron pore size Nucleopore filters (Corning) as described inUren et al., Biochem. Biophys. Res. Comm., 204, 628-634 (1994). Growthfactors or inhibitors were added to the lower chamber, and 32D/c-Metcells washed in serum-free medium were applied to the upper chamber at afinal density of 2×10⁶ cells/ml, and incubated for 8 h at 37° C. Cellsin the lower chamber were counted with an automated cell counter(Coulter, Inc.) and migration was quantitated.

Cultured Cell Lines and cDNA Transfections

The human mammary epithelial cell line 184B5 was maintained in RoswellPark Memorial Institute (RPMI) 1640 medium (Gibco)+10% fetal bovineserum (FBS) and 5 ng/ml epidermal growth factor (Becton Dickinson). Themurine IL-3-dependent cell line 32D was cultured in RPMI 1640+10% FBSand 5% WEHI-3B conditioned medium as a source of IL-3. 32D/c-Met cellswere generated by co-transfection of 32D cells with pMOG human c-MetcDNA and the neomycin-resistance encoding pCEV27 cDNA by electroporationas described in Pierce et al., Science 239, 628-631 (1988). Cells wereselected in G418 and c-Met expression in stable cell lines was detectedby immunoblotting.

The effect of the peptides on the migration of 32D/c-Met cells is shownin FIGS. 2A and 2B. The results are representative of three or moreexperiments. HGF/NK1 stimulated the cell migration in this system almost20-fold over untreated control cells. The peptides 1-3 each reducedHGF/NK1-stimulated cell migration in a dose-dependent manner. The IC50values calculated from these tests were about 1 to about 10 nM.

The effect of the peptides on the migration of human Okajima cells and184B5 mammary epithelial cells is shown in FIGS. 3A and 3B. Results arerepresentative of three or more experiments. In both panels, valuesrepresent the number of migrating cells per unit area on the bottomsurface of the membrane barrier. Mean values from 10 randomly selectedunit areas are calculated from each of three identically-treated wells.Okajima is a highly transformed cell line derived from a human gastriccarcinoma in which the HGF receptor, c-Met, is dramaticallyoverexpressed (approximately 100-fold) relative to normal epithelial HGFtarget cells, such as 184B5. As shown in FIG. 3A, the peptides eachreduced HGF/NK1-stimulated Okajima cell migration in a dose-dependentmanner. The IC50 values calculated from these experiments were in therange of 10 to 30 nM. The same compounds were equally effective inblocking HGF/NK1-stimulated migration by 184B5 cells (FIG. 3B).

HGF exerts a unique and potent effect on the morphology, dispersion, andmovement of Madin-Darby canine kidney (MDCK) epithelial cells known as“scatter” (Stoker et al., supra). In FIG. 4, MDCK cell movement,observed as the dispersion or scatter of single cells from tightlygrouped colonies, was assayed. Photomicrographs show representativeareas from one of triplicate samples for each condition. The resultsreported are representative of three experiments. As shown in FIG. 4,HGF-stimulated MDCK cell scatter was blocked by the peptides at 10 nMeach. The peptides reduced the number of single cells, i.e. those withthe highest level of motility. These data show that the peptidespotently blocked HGF-stimulated migration by both epithelial andhematopoietic cell types, derived from both normal and tumor tissue.

The level of cell migration observed in the absence of HGF stimulationwas not significantly affected by the active mimetics. These datasuggest that these peptides act at the level of HGF regulation of cellmotility, not at the level of the motility apparatus itself. The cellsin all assays remained viable and fully capable of cell divisionfollowing extended (up to 48 hours) treatment with these peptides,confirming that they lack cytotoxic effects.

While the active peptides did not appear to block the HGF-stimulatedspreading of MDCK cells, one of the earliest events in the process ofcell scatter (Ridley et al., Mol. Cell. Biol., 15, 1110-1122 (1995)),they appeared to dramatically reduce the number of single cells, i.e.those with the highest level of motility. Together these data show thatthe mimetics 1-3 potently block HGF-stimulated migration by bothepithelial and hematopoietic cell types, derived from both normal andtumor tissue.

Example 2

This Example illustrates the inhibition of matrix invasion andtubulogenesis by epithelial and endothelial cells, processes that areassociated with angiogenesis.

Materials

The truncated HGF isoform HGF/NK1 was produced in a bacterial expressionsystem, purified and refolded as previously described in Example 1.Peptides 1-4 were synthesized and purified as described in Example 1 andYao et al, supra. Human recombinant basic fibroblast growth factor(bFGF) and human recombinant vascular endothelial growth factor (VEGF)were from R&D Systems (Minneapolis, Minn.).

Cultured Cells Lines

TAC-2 (Soriano et al., J. Cell Science, 108: 413-430 (1995)), a normalmammary gland epithelial cell line, was cultured in high-glucose DMEM(Gibco BRL Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal bovine serum (FBS) (Biofluids, Rockville, Md.). Madin-Darby caninekidnev (MDCK) epithelial cells were maintained in DMEM+10% FBS. Humandermal microvascular (HMEC-1) endothelial cells (Adeset al., J. Invest.Dermatol., 99: 683-690 (1992)) were grown in RPMI 1640 (Bioflulds)containing 10% FBS and 2 mM glutamine. Human microvascular endothelial(HMVE) cells from neonatal dermis were purchased from CascadeBiologicals and cultured in Medium 131, containing with MVGS (mediasupplement) and 1% Glutamine, as indicated by the manufacturer. Humanumbilical vein endothelial (HUVE) cells were isolated from freshlydelivered cords as reported previously (Jaffee et al., J. Clin. Invest.,52: 2745-2756 (1973)) and grown on Nunclon dishes (Nunc, Dem-nark) inRPMI 1640 supplemented with 20% bovine calf serum (Hyclone Laboratories,Logan, Utah), 50 μg/ml gentamycin, 2.5 μg/ml amphotericin B (fungizone)(Life Technologies), 5 U/ml sodium heparin (Fisher Scientific,Pittsburgh, Pa.), and 200 μg/ml endothelial cell growth supplement(ECGS) (Collaborative Research, Bedford, Mass.) and were used betweenpassages 3 and 6.

Extracellular Matrix Invasion Assay

MDCK cell invasion into three-dimensional collagen gels was analyzed aspreviously described. Briefly, type I collagen (1.5 mg/ml; CohesionTechnologies) was mixed with 10×MEM and sodium bicarbonate (11.76 mg/ml)at a ratio of 8:1:1 (vol:vol:vol) on ice, and 0.4 ml aliquots weredispensed into 16-mm tissue culture wells (Nunc), and allowed to gel at37° C. for 20 min. Cells were seeded onto gels (1×10⁴ cells/well) in 0.4ml of growth medium containing HGF and/or peptides 1 or 4 as indicated.After 5 days, cells were fixed in situ in 2.5% glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.4), and cells that had invaded the gelbelow the surface monolayer in ten randomly selected fields (1×1.4 mm)were counted microscopically using a 20× phase contrast objective. Depthof cellular invasion into the collagen gel was quantitated in the sameten fields per treatment group using a calibrated fine focusingmicrometer. Values were compared using the Student's unpaired t-test anda significant value was taken as P<0.001. Results in Table 1 are shownas the mean number of invading cells/field or mean invasion depth/cellin microns+standard error of the mean. TABLE 1 MDCK Cell Invasion intoCollagen Matrices Mean Invading Mean Invasion Treatment GroupCells/Field Depth/Cell (μm) Control 0 0 HGF 29.7 ± 2.6 35.6 ± 2.7 HGF +peptide 2 12.6 ± 1.9 17.9 ± 1.9 HGF + peptide 4 34.3 ± 2.7 30.8 ± 2.9MDCK cells were left untreated (Control), treated with HGF (10 ng/ml),or HGF+peptide 2 or 4 (100 nM), and invasion into collagen matrices wasquantitated microscopically as described above. Values are the mean ofat least 10 randomly selected fields±standard error of the mean.Epithelial Tube Formation Assay

TAC-2 cells were suspended in three-dimensional collagen gels at 1×10⁴cells/ml in collagen and incubated in complete medium containing HGFand/or Grb2 inhibitors as indicated. After 3 days, the cultures werefixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, and at least3 randomly selected fields per experimental condition in each of 3separate experiments were digitally recorded with brightfieldmicroscopy. The total length of the cords in each individual colonypresent in each optical field was measured with IPLab. Cord length wasconsidered as “0” in: a) colonies with a spheroidal shape, and b)slightly elongated structures in which the length to diameter ratio wasless than 2. The mean values for each experimental condition werecompared to controls using the Student's unpaired t-test. Results inFIG. 5 are described as mean total cord length (in μm) per field.

Cell Migration Assays

The migration assay of HUVE and HMEC-1 cells in modified Boyden chamberswas adapted from previously described procedures (Murohara et al.,Thromb. Vasc. Biol., 19: 1156-61 (1999), Malinda et al., FASEB J., 13:53-62 (1999)). In brief, Biocoat Cell Environment control inserts (8micron pore size; Becton Dickinson) were coated with 0.1% gelatin(Sigma) for at least 1 hour at 37° C. and air dried. Lower chamberscontained 0.7 ml RPMI+0.1% BSA, to which 20 ng/ml HGF and/or 300 nM Grb2inhibitors were added. Cells were pre-treated with the indicatedconcentrations of Grb2 inhibitors for 18-24 hours, trypsinized, washedtwice in RPMI+0.1% BSA, added to upper chambers (5×10⁴ cells/well) withor without inhibitors in a final volume of 0.5 ml, and incubated for 4 hat 37° C. Cells on the upper surface of each filter were removed with acotton swab, while cells that had traversed to the bottom surface of thefilter were fixed and stained using Diff-Quik (Dade Diagnostics) andcounted using a 10× objective. Mean values from 4 randomly selectedfields (1×1.4 mm) were calculated for each of triplicate wells perexperimental condition. Shown in FIG. 6 is the ratio of growthfactor-treated to control migrating cells designated on the y-axis as“Migration (Fold Increase)” or as the mean number of cells per opticalfield in FIGS. 8-11.

HUVE or HMVE cells were seeded per triplicate in type I collagen-coated48-well plates (Biocoat) (3000 cells/well) in 500p of completeEGM-Bullet Kit medium (BioWhittaker, Walkersville, Md.), allowed toattach for 4 hr and incubated overnight with or without the indicatedconcentrations of peptide 2. The cultures were rinsed twice inserum-free medium and incubated with EBM medium (BioWhittaker)supplemented with 50 μg/ml heparin, 50 μg/ml ascorbic acid and 10% FCS.After either 4 days (HUVE) or 5 days (HMVE), cells were harvested withtrypsin/EDTA and counted with an hemocytometer. The mean number of cellsper ml was calculated from 3 independent measures for each of triplicatewells per experimental condition and compared to controls using theStudent's unpaired t-test. Results set forth in FIG. 12 are described asthe ratio of growth factor-treated to control proliferating cellsdesignated on the y-axis as “Proliferation (Fold Increase)”.

Endothelial Cell Tubulogenesis Assay on Collagen

HMEC-1 cell tubulogenesis assay on collagen gels was adapted from apreviously described procedure (Montesano et al., Cell, 42: 469-77(1985), Pepper et al., Exp. Cell Res., 204: 356-63 (1993)). Cells wereseeded onto collagen gels cast in 16-mm wells (2.5×10⁴ cells/well) andgrown to confluence in reduced growth medium (5% FCS and 150 μg/mlECGS). At confluence cells were pre-treated with or without Grb2inhibitors for 24 hours, after which time medium was replaced by freshmedium supplemented with or without the indicated concentration ofinhibitors and/or 20 ng/ml HGF. Medium and compounds were changed everyday. After 24 hours, the cultures were fixed in situ, and 5 randomlyselected optical fields per experimental condition in each of threeseparate experiments were digitally recorded under phase contrastmicroscopy using a 20× phase contrast objective, by focusing 20 μmbeneath the surface of the gel. Invasion was quantitated using IPLabsoftware (Scanalytics, Fairfax, Va.) by measuring the total length ofall cellular structures that had penetrated beneath the surfacemonolayer either as apparently single cells or in the form of cellcords. Values were compared using Student's unpaired t-test and asignificant value was taken at P<0.001. Results set forth in FIG. 7 arethe mean total length (in μm) per field.

HUVE Cell Tubulogenesis Assay on Matrigel

The HUVE tube formation assay was performed as previously described(Grant et al., J. Cell. Physiol., 153: 614-625 (1992)). Briefly, 96-wellplates were coated with 90 μl of Matrigel (10 mg/ml) (CollaborativeResearch) (Baatout, supra) and incubated at 37° C. for 30 min to promotegelling. 10,000 HUVECs were resuspended in reduced growth medium (serumconcentration 10% and 5 U/ml heparin) and added to each well with theindicated reagents in a final volume of 100 μl. After 18 h, the plateswere fixed with Diff-Quik, and at least 4 randomly selected fields perexperimental conditions were digitally recorded under bright fieldillumination using a 10× objective. The mean additive length of thecords present in each optical field was measured using IPLab andcompared to controls using the Student's unpaired t-test.

The ability of MDCK cells to invade three-dimensional collagen matrices,a prerequisite for HGF-stimulated branching morphogenesis, was assessedin the presence of peptides 3 and 4 (Table 1). After 5 days in culturein the absence of HGF, MDCK cells remain as a monolayer on the surfaceof the collagen gel, but HGF stimulates a high proportion of these cellsto invade the gel (35 mm mean depth of invasion; Table 1). Peptide 3(100 nM) reduced both the number of invading cells, as well as the meandepth of invasion per cell, by at least 50%, while peptide 4 had nosignificant effects (Table 1). MDCK cell viability throughout the 5-dayculture period was unchanged in the absence or presence of the Grb2 SH2domain antagonists. Together with the results of Example 1, these datademonstrate that Grb2 SH2 domain antagonists inhibit two main biologicaleffects of HGF, namely the induction of cell migration and the invasionof extracellular matrix.

The ability of Grb2 SH2 domain antagonists to abrogate the morphogeneticactivities of HGF was assessed. In a first set of experiments, we usedan in vitro model of ductal morphogenesis in which mammary gland-derivedepithelial (TAC-2) cells grown within a three-dimensional collagen gelare induced to form branching duct-like structures by HGF. When grown incollagen gels under control conditions, TAC-2 cells formed small,irregular cell aggregates. In the presence of 20 ng/ml recombinant humanHGF they gave rise, after 3 days, to long branching tubes. In markedcontrast, co-addition of peptide 1 and HGF to the cultures abrogated theelongation and branching of duct-like structures. A quantitativeanalysis of tube formation demonstrated that peptides 1, 2, and 3significantly (p<0.0001) abrogate HGF-induced elongation of epithelialtubes in a dose-dependent manner, a sub-maximal inhibitory effect beingalready observed with 30 nM of inhibitor, and a maximal effect with 3mM, whereas low affinity binding peptide 4 had detectable effect atthese concentrations (FIG. 5, and data not shown).

The effect of peptide 1 on the chemokinetic response of immortalizedhuman microvascular (HMEC-1) and primary human umbilical veinendothelial cells to angiogenic factors was assessed. It was observedthat this compound (300 nM) did not significantly alter the basal levelsof endothelial cell motility (FIGS. 6A-B, open bars). However, itpractically reverted to basal levels the 5.5- and 3.5-fold increase oncell motility induced by 20 ng/ml HGF on HMEC-1 and HUVECS,respectively. Interestingly, this inhibitory activity does not seemlimited to HGF stimulation, as demonstrated by the complete blockade ofbFGF-induced HUVEC migration by 300 nM of peptide 1 (FIG. 6A-B, closedbars). These results suggest that Grb2 inhibitors may preventendothelial cell motility in response to angiogenic stimuli conveyed bydifferent receptor tyrosine kinases.

To assess whether the blockade of endothelial cell motility by Grb2inhibitors might correlate with a loss of morphogenetic response to anangiogenic factor, two alternative models of in vitro endothelialangiogenesis were used. In the first model, microvascular endothelialcells were seeded onto the surface of a collagen type I gel. Thecultures were treated only after they had reached confluence (approx. 1week later). After a further 5-day incubation, HMEC-1 cells grown undercontrol conditions had discretely invaded the subjacent collagen matrixas single cells. Addition of 20 ng/ml HGF to the cultures induced a6-fold increase in collagen invasion by either single cells or shortcell cords devoid of lumen. Co-addition of peptide 1 (30-300 nM) and HGFto the cultures suppressed collagen invasion induced by HGF, asignificant (p<0.001) decrease in the total length of the cords present20 μm below the surface of the gel already being observed at aconcentration of 30 μM (FIG. 7). Thus, although HGF failed to induce theformation of lumen-containing capillary-like structures by HMEC-1 cells,its stimulatory effect on matrix invasion, a process required duringangiogenesis, was blocked in the presence of peptide 1, a Grb2antagonist.

In the second in vitro model of angiogenesis, HUVECs were seeded onto aMatrigel gel layer, and immediately treated with HGF, HGF/NK1, orHGF/NK1 with peptide 1. Under control conditions, the cells migrated onthe surface of the gel, established contact with each other and, after12-18 hours, formed irregular ridges or cords, a process reminiscent ofthe early steps of angiogenesis. Addition of 20-50 ng/ml HGF (not shown)or 300 ng/ml HGF/NK1 to the cultures resulted in the formation of acontinuous, extensive network of thick cords. In contrast, co-additionof HGF/NK1 and peptide 1 (1 μM) markedly reduced the extent of cordformation. A quantitative analysis of mean length of the cords peroptical field demonstrated a significant (p<0.001) 300% increase inducedby HGF/NK1 (3217.0±166.5 μm in HGF/NK1-treated cultures vs. 1189.5±166μm in controls), and a significant (p=0.001) 30% decrease whenco-addition of peptide 1 and HGF/NK1 was compared to HGF/NK1 alone(3217.0±166.5 μm in HGF/NK1 alone vs. 2502.8±108 μm in HGF/NK1 pluspeptide 1). Similar results, although to a lower extent, were observedwhen HGF or HGF/NK1 were substituted by 50 ng/ml bFGF (data not shown).

Taken together, these results demonstrate that a peptide having cellsignal inhibiting activity and cell motility inhibiting activity, namelyGrb2 SH2 domain antagonists, inhibit the formation of epithelialbranching duct-like structures and alter endothelial capillary-likecords induced by the HGF isoform HGF/NK1.

To further characterize the anti-angiogenic effect of the Grb2antagonists, the effect on human neonatal microvascular (HMVE) cellmigration was assessed in the presence or the absence of increasingconcentrations of either HGF (0-50 ng/ml) or bFGF (0.2-50 ng/ml) andpeptide 2 (300 nM). It was observed that peptide 2 abolished (p<0.001)cell migration induced by HGF (FIG. 8A) and significantly (p<0.001)inhibited the biphasic stimulatory activity of bFGF. Remarkably, theeffect of the optimal concentration of bFGF (5 ng/ml) was abolished by72%. (FIG. 8B). The effect of peptide 2 on the activity of the mostpowerful angiogenic molecule known to date, namely, vascular endothelialgrowth factor (VEGF) was assessed. When incubated in modified Boydenchambers in the presence of VEGF, HMEC-1, HMVE and HUVE cells underwentdifferent, albeit significant, degrees of migration (FIG. 9). However,the addition of peptide 2 significantly (p<0.001) reverted the effect ofVEGF in all the cell lines, although the degree of inhibition of VEGFactivity differed among the endothelial cell lines. Similar results wereobserved with antagonist 1 (FIG. 9 and data not shown). These resultsshow that Grb2 inhibitors prevent endothelial cell motility in responseto angiogenic stimuli conveyed by different angiogenic pathways, andthat this antagonistic activity is not restricted to a single type ofendothelial cell.

To determine whether the effect of Grb2 antagonists is restricted to theinhibition of endogenous angiogenic factors or might also revert theeffect of exogenous pro-angiogenic molecules, the effect of peptide 2 onthe migratory properties of HUVE cells was assessed in the presence ofphorbol myristate acetate (PMA), a potent tumor promoter. In vitro, uponexposure to PMA, both microvascular and macrovascular endothelial cellsundertake a vascular morphogenetic program by invading the surroundingextracellular matrix and subsequently forming extensive network ofcapillary-like tubular structures (Montesano et al., Cell, 42:469-77(1985) and references mentioned therein). Addition of PMA (40 ng/ml) tocultures of HUVE cells in modified Boyden chambers, resulted in a 300%increase of cell migration. This pro-angiogenic activity, which cannotbe mimicked by the maximal concentration (5 ng/ml) of bFGF, wasdramatically reverted to basal levels in the presence of 300 nM peptide2 (FIG. 10).

The effect of Grb2 antagonists on the inhibition of other biologicalproperties of endothelial cells relevant to the process of angiogenesiswas assessed. To understand the activity of peptide 2 during growthfactor-induced endothelial cell proliferation, HUVE and HMVE cells werecultivated on type I collagen-coated wells in partially supplementedendothelial culture (EBM) medium, as described in Material and Methods.Under these stringent culture conditions, HGF (25 ng/ml), VEGF (10ng/ml) and bFGF (5 ng/ml) induced a significant (p<0.0001) increase inendothelial proliferation, as evidenced by a 2.3-, 2- and 4.1-foldincrease, respectively in the mean number of macrovascular HUVE cellsper ml. Similar, significant (p<0.001) increases in cell numbers wereobserved in HMVE cells (FIG. 12, open bars). Addition of Grb2 inhibitorpeptide 2 (30 nM, 300 nM) resulted in a significant, albeit markedlydifferent, inhibition of proliferation in HUVE and HMVE cells. Whereaspeptide 2 inhibited serum-dependent (p<0.001) and bFGF-dependent(p<0.0001) HUVE cell proliferation at concentrations of 30 nM, it failedto significantly (p=0.05) inhibit HGF- VEGF-induced proliferation atthis concentration (FIG. 12, gray bars). Only higher concentrations ofcompound (300 nM) were able to induce significant (p<0.0001) reductionin cell counts (FIG. 12, black bars). However, the behavior ofmicrovascular HMVE was dramatically different. While basically resistant(P=0.02-0.05) to 30 nM of the inhibitor both in the presence and absenceof growth factors, highly significant (p<0.001) inhibition of cellgrowth was only observed with 300 nM of compound (FIG. 12, black bars).These results demonstrate that Grb2 inhibitors elicit differentantagonistic effects on angiogenesis pathways depending both on the typeof endothelial cell and the growth factor.

PDGF is implicated in different biological processes such vascularremodeling, wound healing and cancer (for reviews, see Bornfeldt et al.,Ann N Y Acad. Sci., 766:416-30, 1995; Gendron, Surv. Ophthalmol.,44:184-5, 1999). Addition of 50 ng/ml PDGF-BB to NIH 3T3 fibroblastsincubated in a modified Boyden chamber results in a dramatic, 20-foldincrease in cell migration. When co-added simultaneously to thecultures, the Grb2 antagonist peptide 2 inhibits NIH 3T3 cell migrationin a significant (p<0.001), dose-dependent manner, a 60% reduction inthe mean number of cells per field being already observed withconcentrations as low as 30 nM and a further (FIG. 11). This observationopens a potential therapeutical use of the Grb2 inhibitor compounds inthe treatment of diseases such as cancer, wound healing disorders,vascular complications of diabetes mellitus, vascular nephropathies, anddiseases with occurrence of fibrosis.

Example 3

This Example illustrates the inhibition of cancer metastasis by inaccordance with the present invention.

Reagents and Cell Culture

The Grb2-SH2 domain binding antagonist tested was C-90 (peptide 2).PC-3M-luc-C6 cells (Xenogen Inc.) were derived from PC-3M metastatichuman adenocarcinoma cells by stable transfection with North AmericanFirefly Luciferase; cells were maintained in a humidified 5% CO₂incubator with MEM/EBSS media (Hyclone, Logan, Utah) supplemented with10% fetal bovine serum (FBS, Hyclone) and 100 mM Na-pyruvate (Hyclone),non-essential amino acids (Hyclone), vitamins (Invitrogen) andL-glutamine (Hyclone). B16-luc cells were a gift from the Laboratory ofDermatology (NIH, NCI) and were stably transfected with FireflyLuciferase; cells were maintained in DMEM supplemented with 10% FBS.Full-length purified recombinant human HGF protein was obtained from R&DSystems (294-HG, Minneapolis, Minn.).

Chamber Cell-Migration Assay

After serum deprivation and pre-treatment for 16 hrs, PC3M cells wereassayed in cell culture inserts (BD Biosciences), 8 micron-pore-sizepolyethylene terephthalate membrane with Falcon cell culture inserts(Becton Dickinson). Pre-treated cells were trypsinized and counted; atotal of 1×105 cells in serum free medium (500 microliters) were addedto the upper chamber, and 500 microliters of serum free media with therelative concentration of C90 and HGF were added to the lower chamber.The assay was run for 16 hours in the incubator at 37° C. Then the cellsin the inside of the upper chamber were removed with a cotton swab andcell on the other side of the membrane were fixed and stained usingDiff-Quick (Dade Diagnostics, Deerfield, Ill.). Photographs of threerandom fields were taken and cells were counted by bright fieldmicroscopy using a 10× objective. Mean values from four randomlyselected fields (1×1.4 mm) were calculated for each of triplicate wellsper experimental condition.

ACEA Real-Time Cell Invasion and Migration (RT-CIM™) assay. B16 cellswere serum deprived and pre-treated for 16 hrs, prior to being assayedinside the RT-CIM assay system (ACEA Biosciences). Cells were thentrypsinized and counted; a total of 2×10³ cells in serum free media (100microliters) were added to the upper chamber, and 120 microliters ofserum free media with the relative concentration of C-90 and HGF wereadded to the lower chamber. Prior to plating the cells, membranes werepre-coated for 30 minutes on both sides with fibronectin (1microgram/ml) and washed once with PBS. Migration was then monitored forseveral hours.

The results are shown in FIG. 13A-B and 14A-C. FIG. 13A depictsbrightfield photomicrographs of Diff-Quick stained cells that traversedMatrigel-coated Transwell filters (BD Biosciences) as a measure of B16mouse melanoma cell invasiveness in vitro. The upper panels representthe effect on HGF-treated cells (40 ng/ml, 30 h); lower panels depictthe effect on untreated cells. The left panels represent cells nottreated with the SH2 domain antagonist C-90; the right panels representcells receiving increasing concentrations (nM) of C-90 as indicatedabove. Objective magnification is 4×.

FIG. 13B depicts bar graphs of PC3M cell invasion across Matrigel-coatedTranswell filters in the presence (filled bars) or absence (unfilledbars) of HGF (40 ng/ml, 30 h). Increasing concentrations of C-90 (nM)produces dose-dependent inhibition of PC3M cell invasiveness in vitro.

The two cell lines analyzed for invasiveness in vitro were stablytransfected with luciferase so that bioluminescence imaging could beused to assess C-90 efficacy as an anti-metastatic treatment in vivo.Tumor Xenograft Studies in SCID/beige Mice and In vivo Imaging:Tail VeinModel:

B16-luc cells were pre-treated for 24 hrs with 1 micromolar C90, thentrypsinized, washed in PBS and spun down at 650 rpm to be resuspended inHBSS at the appropriate cell density for injection (1×10⁶ cells).Injection was boosted with 10 micromolar C90. Cell where then injectedin the dorsal tail vein of severe combined immunodeficient (SCID)/beigemice (Taconic Inc., Germantown, N.Y.).

Tumor Xenograft Studies in SCID/beige Mice and In vivo Imaging:Subcutaneous Tumor Model:

PC3M-luc cells were pre-treated for 24 hrs with 1 micromolar C90, thentrypsinized, washed in PBS and spun down at 650 rpm to be resuspended inHBSS at the appropriate cell density for injection (1×10⁶ cells).Injection was boosted with 10 micromolar C-90. Cell where then injectedin the right flank SCID/beige mice (Taconic Inc., Germantown, N.Y.). Onindicated days, mice were injected with 150 mg/kg of D-luciferin(potassium salt, Xenogen Corp., Alameda, Calif.) and placed for imagingin the In vivo Imaging System (Xenogen) with total imaging time of 1 or5 minutes. Total body bioluminescence was quantified by integrating thephotonic flux (photons per second) through a region of interest drawnaround each mouse.

Cells prepared for xenograft injections were pretreated with C-90 for 24h. Cells were injected in to mice (5 per group) by either tail vein(B16-luc, induced metastasis model) or subcutaneously (PC3M-luc,spontaneous metastasis model). Imaging of the PC3M cell metastases inlungs ex vivo (FIG. 14A)) correlated with earlier scans of intactanimals (data not shown). Units are proportional to photons emittedwithin a defined time interval (total flux). C-90 significantly reducedthe metastatic rate relative to untreated control cells (top panel), butdid not affect primary tumor growth, as indicated by either mass (FIG.14B, left side) or volume (FIG. 14B, right side) assessed ex vivo,consistent with inhibition of motility and invasion, but not cellproliferation, in vitro. Uninhibited growth of the primary tumor alsosupports the contention that metastasis was blocked due to poor cellviability of the treated cells, as well as by parallel experiments, inwhich cells treated with C-90 for 24 h show identical growth rate andviability as untreated cells over a the three week xenograft studyperiod.

B16 mouse melanoma cells receiving a single 24 h pre-treatment with C-90also showed a significantly reduced ability to form lung metastases overa three week period relative to untreated control cells (FIG. 14C). Asfor PC3M cells, no differences in cell viability or growth rate inculture were associated with C90 treatment over the same study period.

Data are expressed as mean+/−SD and analyzed using GraphPad Prismsoftware. Mean values among groups were compared for statisticalsignificance using unpaired t-test or ANOVA.

The invention encompasses the following aspects.

1. A method of inhibiting cell motility in a mammal comprisingadministering to said mammal a peptide having cell signal inhibitingactivity and cell motility inhibiting activity, wherein said peptide issubstantially free of cytotoxicity.

2. A method of inhibiting angiogenesis in a mammal comprisingadministering to said mammal a peptide having cell signal inhibitingactivity and cell motility inhibiting activity, wherein said peptide issubstantially free of cytotoxicity.

3. The method of aspect 1 or 2, wherein said peptide is a Grb2-SH2domain mimetic peptide.

4. The method of aspect 1 or 2, wherein said peptide recognizes a pYXNmotif.

5. The method of any of aspects 1-4, wherein said cell motility orangiogenesis is induced by the hepatocyte growth factor (HGF).

6. The method of any of aspects 1-5, wherein cell motility is induced bythe binding of c-Met receptor with the Grb2 protein.

7. The method of any of aspects 1-6, wherein said peptide has theformula I

wherein n is 0 to 15, X is a group that modifies an amino group to anamide, PTI is a bivalent radical of tyrosine, a bivalent radical ofphosphotyrosine, or of a phosphotyrosine mimetic; AA stands for abivalent radical of a natural or unnatural amino acid; and Y is asecondary amino group; or a salt thereof.

8. A method for preventing a mammal from being afflicted, or treating amammal afflicted, by a disease, condition, or state that is mediated bythe binding of an intracellular transducer to a receptor proteintyrosine kinase comprising administering to said mammal a peptide of theformula I

wherein n is 0 to 15, X is a group that modifies an amino group to anamide, PTI is a bivalent radical of tyrosine, a bivalent radical ofphosphotyrosine, or of a phosphotyrosine mimetic; AA stands for abivalent radical of a natural or unnatural amino acid; and Y is asecondary amino group; or a salt thereof.

9. A method for inhibiting the binding of an intracellular transducer toa receptor protein tyrosine kinase comprising contacting (a) a samplecontaining the receptor protein tyrosine kinase, (b) the intracellulartransducer, and (c) the peptide of the formula I

wherein n is 0 to 15, X is a group that modifies an amino group to anamide, PTI is a bivalent radical of tyrosine, a bivalent radical ofphosphotyrosine, or of a phosphotyrosine mimetic; AA stands for abivalent radical of a natural or unnatural amino acid; and Y is asecondary amino group; or a salt thereof, under conditions wherein, inthe absence of the peptide, the receptor protein tyrosine kinase bindsto the intracellular transducer; wherein the contacting results in theinhibition of binding of the intracellular transducer to the receptorprotein tyrosine kinase.

10. A method for detecting the inhibition of binding of an intracellulartransducer to a receptor protein tyrosine kinase comprising: (a)contacting a sample containing the receptor protein tyrosine kinase withthe intracellular transducer, separately, in the presence and absence ofthe peptide of the formula I

wherein n is 0 to 15, X is a group that modifies an amino group to anamide, PTI is a bivalent radical of tyrosine, a bivalent radical ofphosphotyrosine, or of a phosphotyrosine mimetic; AA stands for abivalent radical of a natural or unnatural amino acid; and Y is asecondary amino group; or a salt thereof, under conditions that allowfor binding of the receptor protein tyrosine kinase to the intracellulartransducer in the absence of the peptide; (b) determining that bindinghas occurred between the receptor protein tyrosine kinase and theintracellular transducer; and (c) comparing relative binding levels ofthe receptor protein tyrosine kinase to the intracellular transducer inthe presence and absence of the peptide.

11. The method of any of aspects 7-10, wherein X is oxalyl.

12. The method of aspect 10 or 1, wherein n is 1 to 15, and PTI is abivalent radical of phosphotyrosine or of a phosphotyrosine mimetic.

13. The method of any of aspects 7-12, wherein n is 1 to 4; PTI is abivalent radical of tyrosine or a bivalent radical of phosphotyrosine orof a phosphotyrosine mimetic in the form of a bivalent radical of anamino acid selected from the group consisting ofphosphonomethyl-phenylalanine, phosphono-α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-α-hydroxy)methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxy-phenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which is present in the(D,L)-, D- or L-form;

-(AA)_(n)- is a bivalent radical of a tripeptide of the formula

-(AA¹)-(AA²)-(AA³), wherein -(AA¹)- is selected from the groupconsisting of -Ile-, -Ac₅c-, -Ac₆c-, -Asp-, -Gly-, -Phe-, -Ac₇c-, -Nbo-,-Met-, -Pro-, -β-Ala-, -Gln-, -Glu-, -DHph-, -HPh- and -tLe-; -(AA²)- isselected from the group consisting of -Asn-, -β-Ala-, -Gly-, -Ile-, and-Gln-; and -(AA³)- is selected from the group consisting of -Val-,-β-Ala-, -Gly-, -Gln-, -Asp- and Ac₅c-; a bivalent radical of adipeptide of the formula -(AA¹)-(AA²)- wherein -(AA¹)- and -(AA²)- areas recited above;

or a bivalent radical of an amino acid selected from the amino acidsmentioned above; and

Y is a monosubstituted amino selected from the group consisting of loweralkylamino, octylamino, halonaphthyloxy-lower alkylamino,naphthyloxy-lower alkylamino, phenyl-lower alkylamino, di-phenyl-loweralkylamino, (mono- or di-halo-phenyl)-lower alkylamino,naphthalenyl-lower alkylamino, hydroxy-naphthalenyl-lower alkylamino,phenanthrenyl-lower alkylamino; cycloalkylamino; and cycloalkyl-loweralkylamino;

or a salt thereof.

14. The method of any of aspects 10-13, wherein n is 1 to 4; PTI is abivalent radical of phosphotyrosine or of a phosphotyrosine mimetic inthe form of a bivalent radical of an amino acid selected from the groupconsisting of phosphonomethyl-phenylalanine,phosphono-α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-α-hydroxy)-methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxy-phenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which is present in the(D,L)-, D- or L-form;

-(AA)_(n)- is a bivalent radical of a tripeptide of the formula-(AA¹)-(AA²)-(AA³)- wherein -(AA¹)- is selected from the groupconsisting of -Ile-, -Ac₆c-, -Asp-, -Gly- and -Phe-, -(AA²)- is selectedfrom the group consisting of -Asn-, -β-Ala- and -Gly-; and -(AA³)- isselected from the group consisting of -Val-, -β-Ala-, -Gly-, -Gln-,-Asp- and -Ac₅c-;

a bivalent radical of a dipeptide of the formula -(AA¹)-(AA²)- wherein-(AA¹)- is -Ile- or -AC₆c- and -(AA²)- is -Asn- or -β-Ala-;

or a bivalent radical of the amino acid selected from the amino acidsmentioned above; and

Y is a mono substituted amino group having a substituent selected fromthe group consisting of lower alkyl and aryl-lower alkyl;

or a salt thereof.

15. The method of any of aspects 10-14, wherein n is 1 to 4; PTI is abivalent radical of tyrosine or a bivalent radical of phosphotyrosinemimetic in the form of a bivalent radical of an amino acid selected fromthe group consisting of phosphonomethyl-phenylalanine,phosphono-(α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-α-hydroxy)methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxy-phenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which is present in the(D,L)-, D- or the L-form;

-(AA)_(n)- is a bivalent radical of a tripeptide of the formula-(AA¹)-(AA²)-(AA³)- wherein -(AA¹)- is selected from the groupconsisting of -Ile-, -Ac₅c-, Ac₆c-, -Asp-, -Gly-, -Phe-, -Ac₇c-, -Nbo-,-Met-, -Pro-, -β-Ala-, -Gln-, -Glu-, -DHph-, -HPh- and -tLe-; -(AA²)- isselected from the group consisting of -Asn-, -β-Ala-, -Gly-, -Ile-, and-Gln-; and

-(AA³)- is selected from the group consisting of -Val-, -β-Ala, -Gly-,-Gln-, -Asp- and-Ac₅c-;or

a bivalent radical of an amino acid selected from the amino acidsmentioned above; and

Y is a monosubstituted amino selected from the group consisting of loweralkylamino, octylamino, halonaphthyloxy-lower alkylamino,naphthyloxy-lower alkylamino, phenyl-lower alkylamino, di-phenyl-loweralkylamino, (mono- or di-halo-phenyl)-lower alkylamino,naphthalenyl-lower alkylamino, hydroxy-naphthalenyl-lower alkylamino orphenanthrenyl-lower alkylamino, cycloalkylamino, and cycloalkyl-loweralkylamino; or a salt thereof.

16. The method of any of aspects 7-15, wherein X is a moiety attached tothe nitrogen of PTI and is selected from the group consisting of C₁-C₆alkylcarbonyl, oxalyl, C₁-C₆ alkylaminooxalyl, arylaminooxalyl, arylC₁-C₆ alkylaminooxalyl, C₁-C₆ alkoxyoxalyl, carboxy C₁-C₆ alkylcarbonyl, heterocyclyl carbonyl, heterocyclyl C₁-C₆ alkyl carbonyl, arylC₁-C₆ alkyl heterocyclyl C₁-C₆ alkyl carbonyl, aryloxycarbonyl, and arylC₁-C₆ alkoxycarbonyl.

17. The method of any of aspects 7-16, wherein X is oxalyl.

18. The method of any of aspects 7-17, wherein said peptide is selectedfrom the group consisting ofoxalyl-Pmp-Ile-Asn-NH-(3-naphthalen-1-yl-propyl),oxalyl-Pmp-Ile-Asn-NH-(3-(2-hydroxy-naphthalen-1-yl)-propyl),oxalyl-Pmp-Ile-Asn-NH-(3-naphthalen-2-yl-propyl), andoxalyl-Pmp-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl) wherein “Pmp” standsfor phosphonomethyl phenylalanine.

19. The method of any of aspects 7-10, wherein PTI is a phenylalanylradical having a phenyl ring, an amine end, and a carboxyl end, thephenyl ring having one or more substituents selected from the groupconsisting of hydroxyl, carboxyl, formyl, carboxyalkyl, carboxyalkyloxy,dicarboxyalkyl, dicarboxyalkyloxy, dicarboxyhaloalkyl,dicarboxyhaloalkyloxy, and phosphonoalkyl, phosphonohaloalkyl, whereinthe alkyl portion of the substituents may be unsubstituted orsubstituted with a substituent selected from the group consisting ofhalo, hydroxy, carboxyl, amino, aminoalkyl, alkyl, alkoxy, and keto;

X is a moiety attached to the nitrogen of PTI and is selected from thegroup consisting of alkylcarbonyl, oxalyl, alkylaminooxalyl,arylaminooxalyl, arylalkylaminooxalyl, alkoxyoxalyl, carboxyalkylcarbonyl, heterocyclyl carbonyl, heterocyclylalkyl carbonyl, arylalkylheterocyclylalkyl carbonyl, aryloxycarbonyl, and arylalkoxycarbonyl,wherein the aryl and alkyl portions of the substituents may beunsubstituted or substituted with a substituent selected from the groupconsisting of halo, hydroxy, carboxyl, amino, aminoalkyl, alkyl, alkoxy,and keto; and the heterocyclyl portion of Y contains at least 4 heteroatoms selected from the group consisting of O, N, and S;

AA is an amino acid, the amine end of which is attached to the carboxylend of PTI; and

Y is an arylalkylamino or arylheterocyclyl alkylamino;

or a salt thereof.

20. The method of aspect 19, wherein PTI is a phenylalanyl radicalhaving a phenyl ring, an amine end, and a carboxyl end, the phenyl ringhaving one or more substituents selected from the group consisting ofhydroxyl, carboxyl, formyl, carboxy C₁-C₆ alkyl, carboxy C₁-C₆ alkyloxy,dicarboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkyloxy, dicarboxyhalo C₁-C₆alkyl, dicarboxyhalo C₁-C₆ alkyloxy, and phosphono C₁-C₆ alkyl,phosphonohalo C₁-C₆ alkyl, wherein the alkyl portion of the substituentsmay be unsubstituted or substituted with a Substituent selected from thegroup consisting of halo, hydroxy, carboxyl, amino, aminoalkyl, C₁-C₆alkyl, C₁-C₆ alkoxy, and keto;

X is a moiety attached to the nitrogen of PTI and is selected from thegroup consisting of C₁-C₆ alkylcarbonyl, oxalyl, C₁-C₆ alkylaminooxalyl,arylaminooxalyl, aryl C₁-C₆ alkylaminooxalyl, C₁-C₆ alkoxyoxalyl,carboxy C₁-C₆ alkyl carbonyl, heterocyclyl carbonyl, heterocyclyl C₁-C₆alkyl carbonyl, aryl C₁-C₆ alkyl heterocyclyl C₁-C₆ alkyl carbonyl,aryloxycarbonyl, and aryl C₁-C₆ alkoxycarbonyl, wherein the aryl andalkyl portions of the substituents may be unsubstituted or substitutedwith a substituent selected from the group consisting of halo, hydroxy,carboxyl, amino, amino C₁-C₆ alkyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, and keto;and the heterocyclyl portion of Y contains at least 4 hetero atomsselected from the group consisting of O, N, and S;

AA is an amino acid, the amine end of which is attached to the carboxylend of PTI; and

Y is an aryl C₁-C₆ alkylamino or arylheterocyclyl C₁-C₆ alkylamino;

or a salt thereof.

21. The method of aspect 20, wherein PTI is of the formula II:

wherein D has the formula XII, XIII, or XIV:

wherein R₃ and R₄ may be the same or different and are selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, aryl, aryl C₁-C₆ alkyl, C₁-C₆alkaryl, and heteroaryl; and R₅ and R₆ may be the same or different andare selected from the group consisting of hydrogen, halo, hydroxy,amino, and C₁-C₆ alkoxy; and

E is selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₁-C₆alkylcarbonyl, carboxyl, and C₁-C₆ alkylcarbonyl C₁-C₆ alkyl.

22. The method of any of aspects 19-21, wherein Y is aryl C₁-C₆alkylamino.

23. The method of aspect 22, wherein the aryl portion of Y has theformula:

wherein Q₁ is hydrogen or a substituent selected from the groupconsisting of hydroxyl, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, amino, andC₁-C₆ acylamino.

24. The method of aspect 22, wherein the heterocyclyl portion of Y hasthe formula:

wherein Q₂ is hydrogen or a substituent selected from the groupconsisting of hydroxyl, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, amino, andC₁-C₆ acylamino, and F and G are independently selected from the groupconsisting of C, N, O, and S.

25. The method of any of aspects 20-24, wherein X is selected from thegroup consisting of acetyl, oxalyl, C₁-C₆ alkylaminooxalyl,arylaminooxalyl, aryl C₁-C₆ alkylaminooxalyl, C₁-C₆ alkoxyoxalyl,carboxymethylcarbonyl, tetrazolylcarbonyl, tetrazolylmethylcarbonyl,aminophenylmethoxycarbonyl, amino naphthyloxycarbonyl, andmethoxyphenylmethyl tetrazolylmethylcarbonyl.

26. The method of any of aspects 7-25, wherein n is 1-3.

27. The method of any of aspects 10-26, wherein said peptide is(N-oxalyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl) and(N-acetyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl).

28. The method of any of aspects 8 and 11-27, wherein said disease,state, or condition is a cancer.

29. The method of aspect 28, wherein said cancer is colon cancer.

30. The method of aspect 28, wherein said cancer is breast cancer.

31. The method of aspect 28, wherein said cancer is lung cancer.

32. The method of aspect 28, wherein said cancer is thyroid cancer.

33. The method of aspect 28, wherein said cancer is renal cancer.

34. The method of any of aspects 8 and 11-27, wherein said disease,state, or condition is a sarcoma.

35. The method of any of aspects 8 and 11-27, wherein said disease,state, or condition is glioblastoma.

36. The method of any of aspects 8 and 11-27, wherein said disease,state, or condition is melanoma.

37. The method of any of aspects 8 and 11-27, wherein said disease,state, or condition is lymphoma.

38. The method of any of aspects 8 and 11-27, wherein said disease,state, or condition is leukemia.

39. The method of any of aspects 8 and 11-27, wherein said disease,state, or condition is tumor metastasis.

40. The method of aspect 9 or 10, wherein said method is carried out invitro.

41. The method of aspect 9 or 10, wherein said method is carried out invivo.

42. The method of any of aspects 1-8 and 11-28, wherein the peptideblocks HGF-stimulated cellular matrix invasion.

43. The method of any of aspects 1-8 and 11-28, wherein the peptideblocks HGF-stimulated branching tubulogenesis.

44. The method of any of aspects 1-8 and 11-28, wherein the peptideblocks HGF, VEGF, or bFGF-stimulated migration.

45. The method of any of aspects 1-8 and 11-28, wherein the peptideblocks HGF, VEGF, or bFGF-stimulated cell proliferation.

46. The method of any of aspects 1-8 and 11-28, wherein the peptideblocks HGF, VEGF, or bFGF-stimulated formation of capillary structures.

The references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties.While this invention has been described with an emphasis upon severalembodiments, it will be obvious to those of ordinary skill in the artthat variations of the embodiments may be used and that it is intendedthat the invention may be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the invention as defined bythe following aspects.

1. A method of inhibiting cancer metastasis in an animal in need thereofcomprising administering an effective amount of a compound selected fromthe group consisting of(N-oxalyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl) and(N-acetyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl).
 2. Themethod of claim 1, wherein the cancer is melanoma or prostate cancer. 3.The method of claim 2, wherein the cancer is melanoma.
 4. The method ofclaim 2, wherein the cancer is prostate cancer.
 5. The method of claim1, wherein the compound is(N-oxalyl-4-malonyl)-Phe-Ac₆c-Asn-NH-(3-naphthalen-1-yl-propyl).
 6. Amethod for preventing a mammal from being afflicted, or treating amammal afflicted, by a disease, condition, or state that is mediated bythe binding of an intracellular transducer to a receptor proteintyrosine kinase comprising administering to said mammal a peptide of theformula I

wherein n is 0 to 15, X is a group that modifies an amino group to anamide, PTI is a bivalent radical of tyrosine, a bivalent radical ofphosphotyrosine, or of a phosphotyrosine mimetic; AA stands for abivalent radical of a natural or unnatural amino acid; and Y is asecondary amino group; or a salt thereof.
 7. A method for detecting theinhibition of binding of an intracellular transducer to a receptorprotein tyrosine kinase comprising: (a) contacting a sample containingthe receptor protein tyrosine kinase with the intracellular transducer,separately, in the presence and absence of the peptide of the formula I

wherein n is 0 to 15, X is a group that modifies an amino group to anamide, PTI is a bivalent radical of tyrosine, a bivalent radical ofphosphotyrosine, or of a phosphotyrosine mimetic; AA stands for abivalent radical of a natural or unnatural amino acid; and Y is asecondary amino group; or a salt thereof, under conditions that allowfor binding of the receptor protein tyrosine kinase to the intracellulartransducer in the absence of the peptide; (b) determining that bindinghas occurred between the receptor protein tyrosine kinase and theintracellular transducer; and (c) comparing relative binding levels ofthe receptor protein tyrosine kinase to the intracellular transducer inthe presence and absence of the peptide.
 8. The method of claim 6,wherein n is 1 to 15, and PTI is a bivalent radical of phosphotyrosineor of a phosphotyrosine mimetic.
 9. The method of claim 6, wherein n is1 to 4; PTI is a bivalent radical of phosphotyrosine or of aphosphotyrosine mimetic in the form of a bivalent radical of an aminoacid selected from the group consisting ofphosphonomethyl-phenylalanine, phosphono-α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-(α-hydroxy)-methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxy-phenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which is present in the(D,L)-, D- or L-form; -(AA)_(n)- is a bivalent radical of a tripeptideof the formula -(AA¹)-(AA²)-(AA³)-wherein -(AA1)- is selected from thegroup consisting of -Ile-, -Ac6c-, -Asp-, -Gly- and -Phe-, -(AA²)- isselected from the group consisting of -Asn-, -β-Ala- and -Gly-; and-(AA3)- is selected from the group consisting of -Val-, -β-Ala-, -Gly-,-Gln-, -Asp- and -Ac5c-; a bivalent radical of a dipeptide of theformula -(AA1)-(AA2)- wherein -(AA1)- is -Ile- or -Ac6c- and -(AA2)- is-Asn- or -β-Ala-; or a bivalent radical of the amino acid selected fromthe amino acids mentioned above; and Y is a mono substituted amino grouphaving a substituent selected from the group consisting of lower alkyland aryl-lower alkyl; or a salt thereof.
 11. The method of claim 6,wherein n is 1 to 4; PTI is a bivalent radical of tyrosine or a bivalentradical of phosphotyrosine mimetic in the form of a bivalent radical ofan amino acid selected from the group consisting ofphosphonomethyl-phenylalanine, phosphono-α-fluoro)methyl-phenylalanine,phosphono-(α,α-difluoro)methyl-phenylalanine,phosphono-(α-hydroxy)methyl-phenylalanine, O-sulfo-tyrosine,dicarboxymethoxyphenylalanine, aspartic acid, glutamic acid,phosphoserine and phosphothreonine, each of which is present in the(D,L)-, D- or the L-form; -(AA)_(n)- is a bivalent radical of atripeptide of the formula -(AA1)-(AA2)-(AA3)-wherein -(AA1)- is selectedfrom the group consisting of -Ile-, -Ac5c-, Ac6c-, -Asp-, -Gly--Phe-,-Ac₇c-, -Nbo-, -Met-, -Pro-, -β-Ala-, -Gln-, -Glu-, -DHph-, -HPh- and-tLe-; -(AA²)- is selected from the group consisting of -Asn-, -β-Ala-,-Gly-, -Ile-, and -Gln-; and -(AA3)- is selected from the groupconsisting of -Val-, -β-Ala, -Gly-, -Gln-, -Asp-and-Ac5c-; or a bivalentradical of an amino acid selected from the amino acids mentioned above;and Y is a monosubstituted amino selected from the group consisting oflower alkylamino, octylamino, halonaphthyloxy-lower alkylamino,naphthyloxy-lower alkylamino, phenyl-lower alkylamino, di-phenyl-loweralkylamino, (mono- or di-halo-phenyl)-lower alkylamino,naphthalenyl-lower alkylamino, hydroxynaphthalenyl-lower alkylamino orphenanthrenyl-lower alkylamino, cycloalkylamino, and cycloalkyl-loweralkylamino; or a salt thereof.
 12. The method of claim 6, wherein X isoxalyl.
 13. The method of claim 6, wherein said disease, state, orcondition is a cancer.
 14. The method of claim 13, wherein said canceris colon cancer.
 15. The method of claim 13, wherein said cancer isbreast cancer.
 16. The method of claim 13, wherein said cancer is lungcancer.
 17. The method of claim 13, wherein said cancer is thyroidcancer.
 18. The method of claim 13, wherein said cancer is renal cancer.19. The method of claim 6, wherein said disease, state, or condition isa sarcoma.
 20. The method of claim 6, wherein said disease, state, orcondition is glioblastoma.
 21. The method of claim 6, wherein saiddisease, state, or condition is melanoma.
 22. The method of claim 7,wherein said disease, state, or condition is lymphoma.
 23. The method ofclaim 6, wherein said disease, state, or condition is leukemia.
 24. Themethod of claim 8, wherein said disease, state, or condition is tumormetastasis.